The present invention relates generally to methods of purifying recombinant Stat proteins, modified Stat proteins and functional fragments thereof. Included in the present invention are the purified proteins and fragments themselves. The present invention also relates to methods of separating phosphorylated species of these proteins and fragments from the nonphosphorylated forms. The present invention also relates to methods for using purified Stat proteins, truncated Stat proteins or N-terminal fragments of Stat proteins for drug discovery.
Transcription factors play a major role in cellular function by inducing the transcription of specific mRNAs. Transcription factors, in turn, are controlled by distinct signalling molecules. One particular family of transcription factor consists of the Signal Transducers and Activators of Transcription (Stat) proteins. Presently, there are seven known mammalian Stat family members. The recent discovery of Drosophila and Dictyostelium discoideum Stat proteins suggest that Stat proteins have played an important role in signal transduction-since the early stages of our evolution [Yan R. et al., Cell 84:421-430 (1996); Kawata et al., Cell 89:909 (1997)]. Stat proteins mediate the action of a large group of signalling molecules including the cytokines and growth factors (Darnell et al. WO 95/08629, 1995). One distinctive characteristic of the Stat proteins are their apparent lack of requirement for changes in second messenger, e.g., cAMP or Ca++, concentrations. Another characteristic is that Stat proteins are activated in the cell cytoplasm by phosphorylation on a single tyrosine (Darnell et al., 1994: Schindler and Darnell, 1995). The responsible kinases are either ligand-activated transmembrane receptors with intrinsic tyrosine kinase activity, such as EGF- or PDGF-receptors, or cytokine receptors that lack intrinsic kinase activity but have associated JAK kinases, such as those for interferons and interleukins (Ihle, 1995). When Stat proteins are phosphorylated, they form homo- or heterodimeric structures in which the phosphotyrosine of one partner binds to the SRC homology domain (SH2) of the other. The newly formed dimer then translocates to the nucleus, binds to a palindromic GAS sequence, thereby activating transcription (Shuai et al., 1994; Qureshi et al., 1995; Leung et al., 1996).
Stat proteins serve in the capacity as a direct messengers between the cytokine or growth factor receptor present on the cell surface, and the cell nucleus. However, since each cytokine and growth factor produce a specific cellular effect by activating a distinct set of genes, the means in which such a limited number of Stat proteins mediate this result remains a mystery. Indeed, at least thirty different ligand-receptor complexes signal the nucleus through the seven known mammalian Stat proteins [Darnell et al., Science 277:1630-1635 (1997)].
Clearly there is a need to further study the biochemistry of Stat proteins. Unfortunately current studies are seriously hampered due to the low quantities of purified protein available. Full-length cDNAs for all mammalian Stats have been cloned. In addition, certain Stat proteins have been expressed in baculovirus-infected insect cells using a His tag at the COOH-terminal end and then purified by Ni-affinity chrornatography (Xu, X., et al., note 9 (1996). However, no one has reported the production of milligram quantities of activated Stat protein, nor more importantly, a purification process amenable to scaling up for such quantitative isolations.
To perform the biochemical studies necessary to understand the mechanism of the Stat-mediated signal transduction, and to configure assays useful for the detection of compounds that modulate Stat function, there remains an unfulfilled requirement for the production of large amounts of pure protein. Furthermore, there is a need for a means of specifically phosphorylating the correct tyrosine residue on a Stat protein and then separating the resulting phosphorylated Stat protein from the unphosphorylated form in quantitative yields. In addition, there is a need to produce large quantities of stable, soluble truncated Stat proteins that retain functional activities of the corresponding native Stat protein. Finally, there is a need to develop methods of isolating these functional truncated Stat proteins.
The citation of any reference herein should not be construed as an admission that such reference is available as xe2x80x9cPrior Artxe2x80x9d to the instant application.
The present invention describes recombinant human Stat proteins which are produced in insect cells infected with recombinant baculovirus. Stable truncated forms of these proteins produced in bacteria are also included in the present invention. The present invention also includes labeled recombinant human Stat proteins and truncated Stat proteins. One aspect of this invention includes the purification of large amounts of these recombinant proteins. These isolated Stat proteins can be isolated in either their activated form, i.e., having a phosphorylated tyrosine, or in the nonphosphorylated state, where the corresponding tyrosine residue is not phosphorylated. A related aspect to the invention details the protease sensitivity of Stat proteins and the important consequences of this particular property. The present invention exploits this property and describes a recombinant truncated Stat protein that can be expressed in a bacterial host in large quantities, as a soluble protein that can be readily purified by the teaching of the present invention. The phosphorylated and nonphosphorylated form of the truncated Stat protein can also be individually isolated.
The expression of the truncated protein in a soluble form overcomes earlier failures, where recombinant Stat proteins formed almost exclusively insoluble inclusion bodies. Other potentially active fragments of Stat proteins that contain the DNA binding domain, either form insoluble inclusion bodies or are themselves so susceptible to proteolysis that isolation of the large quantities necessary for biochemical studies are not practical. Thus the present invention teaches for the first time, a soluble recombinant truncated Stat protein, as well as methods of its expression and isolation.
Although the present invention includes all Stat proteins, when specific amino acid residues are identified by number, the number represents the sequential position of that amino acid in the amino acid sequence of Stat1xcex1. Thus, the number denoted for a specified amino acid in Stat1xcex2 and Stat1tc, as used herein, is per its corresponding position in the amino acid sequence of Stat1xcex1.
The present invention includes a truncated Stat protein that can be expressed as a soluble recombinant protein in a bacterial host cell. In preferred embodiments the bacterial host is E. coli, and the soluble truncated Stat protein makes up at least 30% of the total recombinant truncated Stat protein produced. In a more preferred embodiment the soluble truncated Stat protein makes up at least 50% of the total recombinant truncated Stat protein produced. In one embodiment, the truncated Stat protein has an amino acid sequence substantially similar to SEQ ID NO:3. In another embodiment, the truncated Stat protein has an amino acid sequence of SEQ ID NO:3. In preferred embodiments, the truncated Stat protein is purified. In one variation of this type, the purified truncated Stat protein exhibits a single protein band on 7% SDS-PAGE, run under reducing conditions.
The Stat proteins, including the truncated Stat proteins of the present invention are activated when a tyrosine residue of the protein is phosphorylated. In a preferred embodiment of this type, the phosphorylated tyrosine is tyrosine 701 of the Stat1xcex1 amino acid sequence shown in SEQ ID NO:1.
In one embodiment, the purified truncated Stat protein is substantially or completely free of its phosphorylated form. In another embodiment, the purified truncated Stat protein is substantially or completed phosphorylated. In yet a third embodiment, the purified truncated Stat protein is a mixture of the nonphosphorylated and phosphorylated forms.
One embodiment of the present invention is a purified Stat protein that is either substantially or completely free of its corresponding phosphorylated, activated form or in the alternative, is essentially or entirely in the corresponding phosphorylated, activated form. One variation of this embodiment exhibits a single protein band on 7% SDS-PAGE, run under reducing conditions, and has an amino acid sequence substantially similar to SEQ ID NO:1. In another variation the purified Stat protein, exhibits a single protein band on 7% SDS-PAGE, run under reducing conditions, and has an amino acid sequence substantially similar to SEQ ID NO:2. Yet another variation also includes a purified Stat protein that exhibits a single protein band on 7% SDS-PAGE, run under reducing conditions and has an amino acid sequence of SEQ ID NO:1. In still another variation of this embodiment, the purified Stat protein exhibits a single protein band on 7% SDS-PAGE, run under reducing conditions, and has an amino acid sequence of SEQ ID NO:2.
The truncated Stat proteins and purified Stat proteins including the purified truncated Stat proteins of the present invention can have a converted cysteine. The converted cysteine can be of the form of a modified cysteine, such as a cysteine having a blocked thiol group or of an analogue of cysteine such as homocysteine; or of an amino acid replacement for cysteine. In preferred embodiments of this last type, the amino acid replacement for cysteine is an alternative polar neutral amino acid such as glycine, serine, threonine, tyrosine, asparagine, or glutamine. In more preferred embodiments of this type, the alternative polar neutral amino acid is a glycine, a serine, or a threonine. In preferred embodiments containing modified cysteines, the modified cysteine is as an alkylated cysteine, or a cysteine containing a mercurial, or the thiol is oxidized and forms a disulfide bond with a second thiol moiety.
The alkylated cysteines may be alkylated by a variety of alkylating agents including iodoacetate, sodium tetracyanate, 5,5/dithiobis(2-nitrobenzoic acid), 2,2/-dithiobis-(5-nitropyridine) and N-ethyl maleimide (NEM). In preferred embodiments the alkylated cysteines are alkylated by N-ethyl maleimide.
The purified truncated Stat proteins and purified Stat proteins, including the purified truncated Stat proteins of the present invention, can also have more than one converted cysteine. In one embodiment of this type, the Stat protein is Stat1xcex1 or a fragment thereof and has three converted cysteines at Cysteine 155, Cysteine 440, and Cysteine 492 of the Stat1xcex1 amino acid sequence shown in SEQ ID NO:1. The three converted cysteines can take any form as listed above, including each cysteine taking an alternative form. In one such embodiment Cysteine 155 is alkylated, Cysteine 440 is substituted by homocysteine, and Cysteine 492 is substituted by a threonine. In a preferred embodiment, all three converted cysteines are alkylated cysteines. All of these Stat proteins and purified Stat proteins can be purified to exhibit one band on 7% SDS-PAGE, under reducing conditions in either their phosphorylated, activated state or in their corresponding nonphosphdrylated form.
The present invention also includes purified Stat N-terminal peptide fragments. These peptide fragments consist of a protein domain that can be selectively cleaved by mild proteolysis with subtilisin or proteinase K. The N-terminal peptide fragments can form homodimers. As part of a Stat protein, the N-terminal domain serves to enhance the binding of two adjacent Stat dimers to a pair of closely aligned DNA binding sites, i.e., binding sites separated by approximately 10 to 15 base pairs. In a preferred embodiment, the N-terminal peptide fragment has an amino acid sequence substantially similar to that of SEQ ID NO:4. In a more preferred embodiment, the N-terminal peptide fragment has an amino acid sequence of SEQ ID NO:4.
The present invention, also includes antibodies to the truncated Stat protein, and the N-terminal peptide fragment of a Stat protein, as purified from recombinant sources or produced by chemical synthesis, and derivatives or analogs thereof, including fusion proteins. Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and a Fab expression library. These antibodies may be labeled.
The present invention also includes nucleic acids comprising nucleotide sequences that encode a truncated Stat protein. In one embodiment the nucleic acid comprises a nucleotide sequence that encodes a truncated Stat protein having an amino acid sequence that is substantially similar to SEQ ID NO:3. In a related embodiment the nucleic acid comprises a nucleotide sequence that encodes a truncated Stat protein having the amino acid sequence of SEQ ID NO:3. In yet another embodiment the nucleic acid comprises a nucleotide sequence that is substantially similar to SEQ ID NO:5 and codes for the expression of a truncated Stat protein. In still another embodiment the nucleic acid contains a nucleotide sequence having the sequence of SEQ ID NO:5.
The present invention also includes nucleic acids that comprise a nucleotide sequence encoding an N-terminal fragment of a Stat protein. In one embodiment the nucleic acid comprises a nucleotide sequence that encodes a Stat N-terminal fragment having an amino acid sequence that is substantially similar to SEQ ID NO:4. In a related embodiment the nucleic acid comprises a nucleotide sequence that encodes a Stat N-terminal fragment having the amino acid sequence of SEQ ID NO:4. In yet another embodiment the nucleic acid comprises a nucleotide sequence that is substantially similar to SEQ ID NO:6 and codes for the expression of a Stat N-terminal fragment. In still another embodiment the nucleic acid contains a nucleotide sequence having the sequence of SEQ ID NO:6.
All of the nucleic acids of the present invention can also contain heterologous nucleotide sequences.
Methods of phosphorylating the Stat proteins in vitro, are also included in the present invention. In one embodiment the phosphorylation is performed with a preparation of EGF-receptor kinase. In preferred embodiments the EGF-receptor preparation is obtained from cell lysates and purified with the use of an anti-EGF-receptor antibody directed against the extracellular domain. In some such embodiments the resulting EGF-receptor antibody complex is precipitated with Protein A agarose beads. In another preferred embodiment the antibody is a monoclonal antibody. In yet another preferred embodiment the cell lysates are from humans. In the most preferred embodiment of this method, the antibody is a monoclonal antibody and the cell lysates are from humans.
The present invention also includes methods of separating phosphorylated Stat proteins including phosphorylated truncated Stat proteins from their nonphosphorylated counterparts. Although these methods may be properly applied to all Stat proteins, and their corresponding truncated proteins, in preferred embodiments the Stat protein has an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, and the truncated Stat protein has an amino acid sequence substantially similar to SEQ ID NO:3. In more preferred embodiments the Stat protein or the truncated Stat protein also has a converted cysteine. In the most preferred embodiment, the Stat protein or truncated Stat protein has three converted cysteines which are alkylated cysteines at Cysteine 155, Cysteine 440, and Cysteine 492 of the Stat1xcex1 amino acid sequence shown in SEQ ID NO:1.
In one embodiment a mixture containing phosphorylated Stat protein and nonphosphorylated Stat protein are placed onto a heparin-solid support. In preferred embodiments the heparin solid support is either heparin agarose, heparin SEPHADEX or heparin cellulose. In the most preferred embodiment the heparin-solid support is heparin agarose.
In one variation of this embodiment the heparin agarose is washed first with a low-salt buffer to remove materials that either bind more weakly than the nonphosphorylated Stat protein or do not bind at all. The Stat proteins are eluted from the heparin agarose as a function of salt concentration with the nonphosphorylated Stat protein eluting at a lower salt concentration than the phosphorylated protein. In one particular embodiment of this type, the protein is eluted with a salt gradient. In a preferred embodiment, the elution of the heparin agarose is performed stepwise with an approximately 0.15 M monovalent salt elution step, followed by an approximately 0.4 M monovalent salt elution step. In this case the unphosphorylated Stat protein elutes during the first elution step, and the phosphorylated Stat protein elutes during the second elution step. In a more preferred embodiment the monovalent salt is potassium chloride.
This procedure may be performed by a batchwise method, though in preferred embodiments the heparin agarose is placed in a column. The procedure may be performed by simple controlled pumping of the column, or by HPLC, FPLC and any other analogous methodology; or the column may be allowed to flow by the pressure of gravity.
The present invention also includes methods of preparing a purified alkylated Stat protein and methods of preparing a purified alkylated truncated Stat protein. Although these methods may be properly applied to all Stat and truncated Stat proteins, in preferred embodiments the Stat protein has an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, and the truncated Stat protein has an amino acid sequence substantially similar to SEQ ID NO:3. In one such embodiment an expression vector containing a nucleic acid that encodes a Stat protein is placed into a compatible host cell, and the Stat protein is expressed. The compatible host cell is grown, harvested and then the expressed Stat protein is released from the host cell. In a preferred embodiment the expressed Stat protein is released from the host cell by lysing the cells. The Stat protein is then treated with an alkylating agent to alkylate one or more cysteines involved in intersubunit aggregation. The alkylated Stat protein is then isolated, yielding a purified alkylated Stat protein.
In another such embodiment, the expression vector contains a nucleic acid that encodes a truncated Stat protein. The truncated Stat protein has an amino acid sequence having an N-terminal sequence that is substantially similar to the N-terminus of the corresponding resulting Stat protein following the cleavage of the proteolytic sensitive N-terminal domain from the corresponding Stat protein. The carboxyl terminus of the truncated Stat protein extends at least to the phosphorylatable tyrosine required for homodimerization. In preferred embodiments, alkylation is performed by incubating the Stat protein with N-ethyl maleimide. In more preferred embodiments, about 40 to 50 mg of purified alkylated truncated Stat protein can be obtained from 6 liters of starting culture. These methods can also include a step of phosphorylating the Stat protein either prior to or preferably following alkylation. In preferred methods of this type, preparations of EGF-receptor kinase are used in the in vitro phosphorylating step.
The present invention also includes methods of preparing a purified substituted Stat protein including methods of preparing a purified substituted truncated Stat protein. Although these methods may be properly applied to all Stat proteins including truncated Stat proteins, in preferred embodiments the Stat protein has an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, and the truncated Stat protein has an amino acid sequence substantially similar to SEQ ID NO:3. In one such embodiment, an expression vector contains a nucleic acid that encodes a substituted Stat protein that has an alternative amino acid substituted for a cysteine of the Stat protein, thereby replacing it. In one preferred embodiment, the amino acid is a polar neutral amino acid. In a variation of this embodiment the alternative polar neutral amino acid is a glycine. In another variation of this embodiment, the alternative polar neutral amino acid is a serine. In still another variation of this embodiment, the alternative polar neutral amino acid is a threonine. In preferred embodiments, the cysteine that has been replaced was involved in the intersubunit aggregation that takes place between Stat proteins.
The expression vector is then placed into a compatible host cell, and the substituted Stat protein is expressed. The compatible host cell is grown, harvested and then the expressed substituted Stat protein is released from the host cell. In a preferred embodiment the expressed Stat protein is released from the host cell by lysing the cells. The substituted Stat protein is then isolated, yielding a purified substituted Stat protein.
In one embodiment, the expression vector contains a nucleic acid that encodes a substituted truncated Stat protein. In one such embodiment, an expression vector contains a nucleic acid that encodes a substituted truncated Stat protein that has an alternative polar neutral amino acid substituted for a cysteine of the Stat protein, thereby replacing it. In one variation of this embodiment, the alternative polar neutral amino acid is a glycine. In another variation of this embodiment, the alternative polar neutral amino acid is a serine. In yet another variation of this embodiment, the alternative polar neutral amino acid is a threonine. In a preferred embodiment, the cysteine that has been replaced was involved in the intersubunit aggregation that takes place between Stat proteins. The substituted truncated Stat protein has an amino acid sequence which is essentially the same as the protease-resistant domain of the Stat protein. In preferred embodiments, about 40 to 50 mg of purified substituted truncated Stat protein can be obtained from 6 liters of starting culture. These methods can also include a step of phosphorylating the Stat protein or truncated Stat protein. In a preferred methods of this type, an EGF-receptor kinase preparation is used in the in vitro phosphorylating step.
In some embodiments, a substituted Stat protein or a substituted truncated Stat protein is also alkylated. In such cases an expression vector containing a nucleic acid that encodes a substituted Stat protein or a substituted truncated Stat protein is placed into a compatible host cell, and expressed. In one embodiment the substituted Stat protein contains a replacement amino acid that is an alternative polar neutral amino acid. In a preferred embodiment the alternative polar neutral amino acid is a glycine, a serine, or a threonine. The compatible host cell is grown, harvested and then the expressed substituted Stat protein or substituted truncated Stat protein is released from the host cell as described herein. The substituted Stat protein or substituted truncated Stat protein is then treated with an alkylating agent to alkylate one or more cysteines involved in intersubunit aggregation. The alkylated substituted Stat protein or alkylated substituted truncated Stat protein is then isolated, yielding a purified alkylated substituted Stat protein or purified alkylated substituted truncated Stat protein. In preferred embodiments, alkylation is performed by incubating the Stat protein or truncated Stat protein with N-ethyl maleimide. In more preferred embodiments about 40 to 50 mg of purified alkylated substituted truncated Stat protein can be obtained from 6 liters of starting culture.
The present invention also includes methods of identifying drugs that effect the interaction of N-terminal domains of Stat proteins that are bound to adjacent DNA binding sites. In one such embodiment, a drug library is screened by assaying the binding activity of a Stat protein to its DNA binding site. This assay is based on the ability of the N-terminal domain of Stat proteins to substantially enhance the binding affinity of two adjacent Stat dimers to a pair of closely aligned DNA binding sites, i.e., binding sites separated by approximately 10 to 15 base pairs. Such drug libraries include phage libraries as described below, chemical libraries compiled by the major drug manufacturers, mixed libraries, and the like. Any of such compounds contained in the drug libraries are suitable for testing as a prospective drug in the assays described below, and further in a high throughput assay based on the methods described below.
One such embodiment includes a method of identifying a drug that interferes with the By interaction of the N-terminal domains of Stat proteins bound to DNA binding sites. One variation of this embodiment relies on a truncated Stat protein that is missing the N-terminal domain responsible for enhancing the binding of two adjacent Stat dimers to a pair of closely aligned DNA binding sites. The binding affinity of a Stat protein to a DNA binding site effected by the N-terminal interaction of Stat proteins is determined. The effect of a prospective drug on the affinity of the Stat protein-DNA binding is determined. If the prospective drug decreases the binding affinity of the Stat protein to a DNA binding site, it becomes a candidate drug. The binding affinity of the corresponding truncated Stat protein to that DNA binding site is also determined. The effect of a candidate drug on the affinity of the truncated Stat protein-DNA binding is determined. If the candidate drug has no effect on the truncated Stat protein-DNA binding, then it can be concluded that the candidate drug interferes with the interaction of N-terminal domains of Stat proteins bound to adjacent DNA binding sites. In a preferred embodiment, the truncated Stat protein has an amino acid sequence that is substantially similar to SEQ ID NO:3.
This variation also includes a method of identifying a drug that enhances the interaction of the N-terminal domains of Stat proteins bound to DNA binding sites. The binding affinity of a Stat protein to a DNA binding site effected by the N-terminal interaction of Stat proteins is determined. The effect of a prospective drug on the affinity of the Stat protein-DNA binding is determined. If the prospective drug increases the binding affinity of the Stat protein to a DNA binding site, it becomes a candidate drug. The binding affinity of the corresponding truncated Stat protein to that DNA binding site is also determined. The effect of a candidate drug on the affinity of the truncated Stat protein-DNA binding is determined. If the candidate drug has no effect on the truncated Stat protein-DNA binding, then it can be concluded that the candidate drug enhances the interaction of N-terminal domains of Stat proteins bound to adjacent DNA binding sites. In a preferred embodiment, the truncated Stat protein has an amino acid sequence that is substantially similar to SEQ ID NO:3.
In another embodiment, a drug library is screened by assaying the binding activity of the two N-terminal fragments of the present invention. As disclosed in the present invention, the N-terminal fragments of Stat proteins form stable dimers in solution. These dimers could mimic the role the N-terminal domain plays in the native Stat protein. Therefore, a prospective drug capable of disrupting or enhancing the stability of the dimer formed between two N-terminal fragments becomes a candidate for a drug capable of destabilizing or stabilizing respectively, N-terminal domain-dependent Stat-DNA binding. These candidate drugs then can be tested in an in vitro or in vivo assay with Stat proteins. For example, dimerization of the N-terminal fragments in solution can be determined using techniques such as fluorescence depolarization.
In yet another embodiment, an N-terminal fragment of a Stat protein is attached to a solid support. The solid support is washed to remove unreacted species. A solution of free N-terminal fragments is poured onto the solid support and the N-terminal fragments are allowed to form dimers with their bound counterparts. In one variation, the solid support is washed again to remove N-terminal fragments that do not bind. Prospective drugs can be screened for their ability to disrupt the dimers, or the formation of the dimers, and thereby increase the concentration of free N-terminal fragments. In a variation of this embodiment, prospective drugs may be screened that enhance the binding of the free N-terminal fragments with their bound counterparts. In this case, there is a corresponding decrease in the concentration free N-terminal fragments. In either case, the measurement of an equilibrium constant, or a dissociation rate constant or an off-rate, may be used to express the effect of the prospective drug on the N-terminal fragment dimer binding. In another variation of this embodiment, prospective drugs that modulate the interaction of the N-terminal domain can be screened by determining the amount of N-terminal fragment that remains bound in the presence of the prospective drug. As compared to the amount of bound fragment in the absence of a prospective drug, prospective drugs that disrupt the interaction result in lower levels of bound fragments, whereas prospective drugs which enhance the interaction result in higher levels of bound fragment. One method of monitoring such interactions is through the use of free N-terminal fragments which have been labeled. Some suitable labels are exemplified below. Alternatively, the dimerization of the free N-terminal fragments with the bound N-terminal fragments can be monitored by changes in surface plasmon resonance. In preferred embodiments the N-terminal fragment has an amino acid sequence substantially similar to SEQ ID NO:4.
In yet another embodiment, the affect of a prospective drug (a test compound) on interactions between N-terminal domains of STATs is assayed in living cells that contain or can be induced to contain activated STAT proteins, i.e., STAT protein dimers. Cells containing a reporter gene, such as the heterologous gene for luciferase, green fluorescent protein, chloramphenicol acetyl transferase or xcex2-galactosidase, operably linked to a promoter comprising two weak STAT binding sites are contacted with a prospective drug in the presence of a cytokine which activates the STAT(s) of interest. The amount (and/or activity) of reporter produced in the absence and presence of prospective drug is determined and compared. Prospective drugs which reduce the amount (and/or activity) of reporter produced are candidate antagonists of the N-terminal interaction, whereas prospective drugs which increase the amount (and/or activity) of reporter produced are candidate agonists. Cells containing a reporter gene operably linked to a promoter comprising strong STAT binding sites are then contacted with these candidate drugs, in the presence of a cytokine which activates the STAT(s) of interest. The amount (and/or activity) of reporter produced in the presence and absence of candidate drugs is determined and compared. Drugs which disrupt interactions between the N-terminal domains of the STATs will not reduce reporter activity in this second step. Similarly, candidate drugs which enhance interactions between N-terminal domains of STATs will not increase reporter activity in this second step.
In an analogous embodiment, two reporter genes each operably under the control of one of the two types promoters described above can be comprised in a single host cell as long as the expression of the two reporter gene products can be distinguished. For example, different modified forms of green fluorescent protein can be used as described in U.S. Pat. No. 5,625,048, Issued Apr. 29, 1997, hereby incorporated by reference in its entirety.
Antagonists of the STAT N-terminal interaction would be expected to antagonize aspects of STAT function. Such candidate drugs are expected to be useful for the treatment of a variety of disease states, including but not limited to, inflammation, allergy, asthma, and leukemias. Candidate drugs which stabilize the N-terminal interaction would be expected to enhance STAT function, and may therefore have utility in the treatment of anemias, neutropenias, thrombocytopenia, cancer, obesity, viral diseases and growth retardation, or other diseases characterized by a insufficient STAT activity.